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1.
Nat Commun ; 12(1): 5243, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475406

RESUMO

Peroxisome, a special cytoplasmic organelle, possesses one or more kinds of oxidases for hydrogen peroxide (H2O2) production and catalase for H2O2 degradation, which serves as an intracellular H2O2 regulator to degrade toxic peroxides to water. Inspired by this biochemical pathway, we demonstrate the reactive oxygen species (ROS) induced tumor therapy by integrating lactate oxidase (LOx) and catalase (CAT) into Fe3O4 nanoparticle/indocyanine green (ICG) co-loaded hybrid nanogels (designated as FIGs-LC). Based on the O2 redistribution and H2O2 activation by cascading LOx and CAT catalytic metabolic regulation, hydroxyl radical (·OH) and singlet oxygen (1O2) production can be modulated for glutathione (GSH)-activated chemodynamic therapy (CDT) and NIR-triggered photodynamic therapy (PDT), by manipulating the ratio of LOx and CAT to catalyze endogenous lactate to produce H2O2 and further cascade decomposing H2O2 into O2. The regulation reactions of FIGs-LC significantly elevate the intracellular ROS level and cause fatal damage to cancer cells inducing the effective inhibition of tumor growth. Such enzyme complex loaded hybrid nanogel present potential for biomedical ROS regulation, especially for the tumors with different redox state, size, and subcutaneous depth.


Assuntos
Antineoplásicos/farmacologia , Nanogéis/química , Peroxissomos/enzimologia , Fotoquimioterapia/métodos , Animais , Antineoplásicos/química , Catalase/química , Catalase/metabolismo , Catálise , Linhagem Celular Tumoral , Óxido Ferroso-Férrico/química , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Verde de Indocianina/química , Camundongos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/efeitos dos fármacos
2.
Science ; 373(6556): 774-779, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385392

RESUMO

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.


Assuntos
Lycopersicon esculentum/parasitologia , Oxigenases de Função Mista/metabolismo , Pectinas/metabolismo , Phytophthora infestans/enzimologia , Doenças das Plantas/parasitologia , Solanum tuberosum/parasitologia , Cobre , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Oxirredução , Phytophthora infestans/genética , Phytophthora infestans/patogenicidade , Folhas de Planta/parasitologia , Polissacarídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361039

RESUMO

Plant G proteins are versatile components of transmembrane signaling transduction pathways. The deficient mutant of heterotrimeric G protein leads to defects in plant growth and development, suggesting that it regulates the GA pathway in Arabidopsis. However, the molecular mechanism of G protein regulation of the GA pathway is not understood in plants. In this study, two G protein ß subunit (AGB1) mutants, agb1-2 and N692967, were dwarfed after exogenous application of GA3. AGB1 interacts with the DNA-binding domain MYB62, a GA pathway suppressor. Transgenic plants were obtained through overexpression of MYB62 in two backgrounds including the wild-type (MYB62/WT Col-0) and agb1 mutants (MYB62/agb1) in Arabidopsis. Genetic analysis showed that under GA3 treatment, the height of the transgenic plants MYB62/WT and MYB62/agb1 was lower than that of WT. The height of MYB62/agb1 plants was closer to MYB62/WT plants and higher than that of mutants agb1-2 and N692967, suggesting that MYB62 is downstream of AGB1 in the GA pathway. qRT-PCR and competitive DNA binding assays indicated that MYB62 can bind MYB elements in the promoter of GA2ox7, a GA degradation gene, to activate GA2ox7 transcription. AGB1 affected binding of MYB62 on the promoter of GA2ox7, thereby negatively regulating th eactivity of MYB62.


Assuntos
Proteínas de Arabidopsis/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Giberelinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Sítios de Ligação , Subunidades beta da Proteína de Ligação ao GTP/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
4.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208368

RESUMO

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.


Assuntos
Alcaloides Indólicos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases/metabolismo , Quinolinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Vimblastina/análogos & derivados , Vias Biossintéticas , Vimblastina/biossíntese , Vimblastina/química
5.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200345

RESUMO

Efficient accumulation of flavonoids is important for increased tolerance to biotic stress. Although several plant defense mechanisms are known, the roles of many pathways, proteins, and secondary metabolites in stress tolerance are unknown. We generated a flavanone 3-hydroxylase (F3H) overexpressor rice line and inoculated Xanthomonas Oryzae pv. oryzae and compared the control and wildtype inoculated plants. In addition to promoting plant growth and developmental maintenance, the overexpression of F3H increased the accumulation of flavonoids and increased tolerance to bacterial leaf blight (BLB) stress. Moreover, leaf lesion length was higher in the infected wildtype plants compared with infected transgenics. Kaempferol and quercetin, which scavenge reactive oxygen species, overaccumulated in transgenic lines compared with wildtypes in response to pathogenic infection, detected by scanning electron microscopy and spectrophotometry. The induction of F3H altered the antioxidant system and reduced the levels of glutathione peroxidase activity and malondialdehyde (MDA) contents in the transgenic lines compared with the wildtypes. Downstream gene regulation analysis showed that the expression of F3H increased the regulation of flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and slender rice mutant (SLR1) during BLB stress. The analysis of SA and JA signaling revealed an antagonistic interaction between both hormones and that F3H induction significantly promoted SA and inhibited JA accumulation in the transgenic lines. SA-dependent nonexpressor pathogenesis-related (NPR1) and Xa1 showed significant upregulation in the infected transgenic lines compared with the infected control and wildtype lines. Thus, the overexpression of F3H was essential for increasing BLB stress tolerance.


Assuntos
Antioxidantes/metabolismo , Resistência à Doença/imunologia , Flavonoides/metabolismo , Hormônios/metabolismo , Oxigenases de Função Mista/metabolismo , Oryza/imunologia , Doenças das Plantas/imunologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Oryza/genética , Oryza/metabolismo , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Estresse Fisiológico , Xanthomonas/fisiologia
6.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065782

RESUMO

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10ß-hydroxylase (T10ßOH) and a newly putative ß-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.


Assuntos
Aciltransferases/metabolismo , Antineoplásicos/metabolismo , Paclitaxel/biossíntese , Extratos Vegetais/biossíntese , Taxus/química , Taxus/enzimologia , Acilação , Aciltransferases/genética , Sequência de Aminoácidos , Vias Biossintéticas , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Ligases/metabolismo , Oxigenases de Função Mista/metabolismo , Taxoides/metabolismo , Taxus/classificação , Taxus/genética , Transcriptoma
7.
Commun Biol ; 4(1): 727, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117349

RESUMO

Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.


Assuntos
Arabidopsis/imunologia , Botrytis/imunologia , Celulose/metabolismo , Oxigenases de Função Mista/metabolismo , Oligossacarídeos/metabolismo , Doenças das Plantas/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Resistência à Doença , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/fisiologia , Oligossacarídeos/fisiologia , Doenças das Plantas/microbiologia , Sordariales/metabolismo
8.
Genes (Basel) ; 12(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067657

RESUMO

We evaluated a transcriptome using high-throughput Illumina HiSeq sequencing and related it to the morphology, leaf anatomy, and physiological parameters of Carpinus putoensis putoensis under NO2 stress. The molecular mechanism of the C. putoensis NO2 stress response was evaluated using sequencing data. NO2 stress adversely affected the morphology, leaf anatomy, and total peroxidase (POD) activity. Through RNA-seq analysis, we used NCBI to compare the transcripts with nine databases and obtained their functional annotations. We annotated up to 2255 million clean Illumina paired-end RNA-seq reads, and 250,200 unigene sequences were assembled based on the resulting transcriptome data. More than 89% of the C. putoensis transcripts were functionally annotated. Under NO2 stress, 1119 genes were upregulated and 1240 were downregulated. According to the KEGG pathway and GO analyses, photosynthesis, chloroplasts, plastids, and the stimulus response are related to NO2 stress. Additionally, NO2 stress changed the expression of POD families, and the HPL2, HPL1, and POD genes exhibited high expression. The transcriptome analysis of C. putoensis leaves under NO2 stress supplies a reference for studying the molecular mechanism of C. putoensis resistance to NO2 stress. The given transcriptome data represent a valuable resource for studies on plant genes, which will contribute towards genome annotations during future genome projects.


Assuntos
Betulaceae/genética , Dióxido de Nitrogênio/metabolismo , Estresse Fisiológico , Transcriptoma , Betulaceae/efeitos dos fármacos , Betulaceae/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dióxido de Nitrogênio/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
9.
Arch Insect Biochem Physiol ; 107(4): e21825, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34164848

RESUMO

Mating triggers physiological and behavioral changes in female insects. In many species, females experience postmating behavioral and physiological changes that define a post-mated state. These changes are comprised of several conditions, including long-term refractoriness to re-mating and increased production and laying of eggs. Here, we report that mating led to several changes in brown planthopper (BPH) females, including increased octopamine (OA), cAMP concentrations, and activities of several enzymes. Mating also led to changes in the expression of several genes acting in female physiology, including those in the cAMP/PKA signal transduction pathway. OA injections into virgin females led to similar changes. RNAi silencing of the gene encoding tyramine ß-hydroxylase, involved in the final step in OA synthesis, led to decreased expression of these genes, and reduced the cAMP/PKA signaling. At the whole-organism level, the RNAi treatments led to reduced fecundity, body weights, and longevity. RNAi silencing of genes acting in OA signaling led to truncated ovarian development, egg maturation, and ovarian vitellogenin (Vg) uptake. The impact of these decreases is also registered at the population level, seen as decreased population growth. We infer that OA signaling modulates the postmating state in female BPH and possibly other hemipterans.


Assuntos
Hemípteros/fisiologia , Oxigenases de Função Mista/metabolismo , Octopamina/metabolismo , Comportamento Sexual Animal/fisiologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Longevidade , Ovário/crescimento & desenvolvimento , Oviposição
10.
Molecules ; 26(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069009

RESUMO

Flavonoids are important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L-1, an induction temperature of 28 °C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caffeic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Flavonoides/biossíntese , Oxigenases de Função Mista/metabolismo , Biocatálise , Cromatografia Líquida de Alta Pressão , Escherichia coli/crescimento & desenvolvimento , Engenharia Genética , Hidroxilação , Especificidade por Substrato , Temperatura , Fatores de Tempo
11.
ACS Chem Biol ; 16(6): 1079-1089, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34032403

RESUMO

Lysobacter are new biocontrol agents known for their prolific production of lytic enzymes and bioactive metabolites. L. enzymogenes is a predator of fungi and produces several structurally distinct antimicrobial compounds, such as the antifungal HSAF (heat stable antifungal factor) and analogs. The mechanism by which L. enzymogenes interacts with fungal prey is not well understood. Here, we found that the production of HSAF and analogs in L. enzymogenes OH11 was significantly induced in media supplemented with ground fungal mycelia or chitin. In the OH11 genome, we identified a gene (LeLPMO10A) that was annotated to encode a chitin-binding protein. The stimulation of HSAF and analogs by chitin was diminished when LeLPMO10A was deleted. We expressed the gene in E. coli and demonstrated that purified LeLPMO10A oxidatively cleaved chitin into oligomeric products, including 1,5 δ-lactones and aldonic acids. The results revealed that LeLPMO10A encodes a lytic polysaccharide monooxygenase, which has not been reported in Lysobacter. The metabolite analysis, antifungal assay, and proteomic analysis showed that the antifungal compounds and the chitin-cleaving LeLPMO10A are colocalized in outer membrane vesicles. The enzymatic products that resulted from in vitro LeLPMO10A-cleaved chitin also significantly induced HSAF and analogs in OH11. Scanning electron microscopic analysis indicated that spherical vesicles were formed outside of OH11 cells, and fewer OH11 cells were observed to attach to fungal hyphae when LeLPMO10A was deleted. Together, the study revealed a previously uncharacterized synergistic strategy utilized by the predatory Lysobacter during interaction with fungal prey.


Assuntos
Antifúngicos/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/metabolismo , Agentes de Controle Biológico/metabolismo , Lysobacter/fisiologia , Oxigenases de Função Mista/metabolismo , Quitina/metabolismo , Fungos/fisiologia , Controle Biológico de Vetores , Polissacarídeos/metabolismo
12.
PLoS Biol ; 19(5): e3001228, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33970909

RESUMO

The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-ß-hydroxylase (tßh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null tßhnM18 allele in 2 behavioral measures in Buridan's paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tßh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tßh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.


Assuntos
Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Alelos , Animais , Animais Geneticamente Modificados/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Genótipo , Masculino , Mutação/genética , Octopamina/genética , Octopamina/metabolismo , Fenótipo , Receptores de Amina Biogênica/genética , Receptores de Amina Biogênica/metabolismo , Tiramina/metabolismo
13.
J Biol Chem ; 297(1): 100823, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34029591

RESUMO

Bacterial two-component flavin-dependent monooxygenases cleave the stable C-S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS-) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C-S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS- closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD's functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Multimerização Proteica , Pseudomonas fluorescens/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Mononucleotídeo de Flavina/metabolismo , Mesilatos/metabolismo , Modelos Moleculares , Especificidade por Substrato , Enxofre/metabolismo
14.
Methods Mol Biol ; 2272: 265-280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009620

RESUMO

TET family enzymes normally oxidize 5-methylcytosine (5mC) in DNA, and play critical roles in shaping the epigenome. Despite their importance, assessing TET activity can be difficult, particularly given the challenge of studying modifications to single nucleobases within complex DNA substrates. We recently demonstrated that in addition to acting on 5mC, TET enzymes can act promiscuously on unnatural nucleobases. Here, we describe how these alternative unnatural substrates can be employed in facile assays to detect and quantify TET activity. DNA containing unnatural 5-vinylcytosine (vC) can be used as a direct endpoint reporter of TET activity, a method that can potentially be adapted to high-throughput platforms. Complementarily, DNA containing unnatural 5-ethynylcytosine (eyC) can trap and inactivate TET enzymes upon reaction, a strategy that can be used to extract active TET enzymes from a complex cellular milieu. We present a detailed PCR-based protocol to synthesize DNA probes with either natural or unnatural modifications, and methods for using these probes to track TET activity either in vitro or in cell extracts.


Assuntos
5-Metilcitosina/metabolismo , Citosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Citosina/metabolismo , Metilação de DNA , Humanos , Especificidade por Substrato
15.
Commun Biol ; 4(1): 562, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980981

RESUMO

Fungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once-enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.


Assuntos
Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/metabolismo , Engenharia de Proteínas/métodos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética
16.
Cancer Sci ; 112(7): 2855-2869, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33970549

RESUMO

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.


Assuntos
Proteína HMGA2/genética , Neoplasias Hepáticas/genética , Oxigenases de Função Mista/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Citosina/metabolismo , Metilação de DNA , Dioxigenases/metabolismo , Epigênese Genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGA2/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxigenases de Função Mista/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regulação para Cima
17.
Methods Mol Biol ; 2272: 195-206, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009615

RESUMO

Aberrant promoter hypermethylation leads to gene silencing and is associated with various pathologies including cancer and organ fibrosis. Active DNA demethylation is mediated by TET enzymes: TET1, TET2, and TET3, which convert 5-methylcytosine to 5-hydroxymethylcytosine. Induction of gene-specific hydroxymethylation via CRISPR/Cas9 gene technology provides an opportunity to reactivate a single target gene silenced in pathological conditions. We utilized a spCas9 variant fused with TET3 catalytic domain to mediate gene-specific hydroxymethylation with subsequent gene reactivation which holds promise for gene therapy. Here, we present guidelines for gene-specific hydroxymethylation targeting with a specific focus on designing sgRNA and functional assessments in vitro.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Sistemas CRISPR-Cas , Metilação de DNA , DNA/análise , Edição de Genes , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Cromatina , Biologia Computacional/métodos , DNA/química , DNA/genética , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Oxirredução , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Sulfitos/química
18.
Nat Commun ; 12(1): 2610, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972531

RESUMO

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Neovascularização Patológica/metabolismo , Proteínas Nucleares/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Cateninas/metabolismo , Movimento Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Transdução de Sinais/genética , Esferoides Celulares/metabolismo
19.
Methods Mol Biol ; 2272: 65-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009609

RESUMO

The enzyme-linked immunosorbent assay (ELISA) technique has been developed half a century ago, and yet its role in molecular biology remains significant. Among the most sensitive of immunoassays, it offers high throughput, combined with affordability and ease of use. This chapter provides the procedure of a highly reproducible indirect sandwich ELISA protocol, which can be applied to a variety of semi-quantitative assays for the investigation of the molecular biology of 5-hydroxymethylcytosine (5hmC) or TET enzymes. Three variations of this protocol are described: assessment and validation of 5hmC-binding proteins, screening and validation of anti-5hmC antibodies, or a readout of TET catalytic activity in in vitro experiments. The assay principle is based on the use of a high affinity avidin-biotin system for efficient immobilization of DNA fragments for further detection by high specificity antibodies. A colorimetric enzymatic reaction is ultimately developed with intensity correlating with the amount of attached antigen.


Assuntos
5-Metilcitosina/análogos & derivados , Avidina/química , Biotina/química , Metilação de DNA , DNA/análise , Ensaio de Imunoadsorção Enzimática/métodos , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/química , 5-Metilcitosina/imunologia , Anticorpos/imunologia , DNA/química , DNA/genética , Genômica , Humanos
20.
Methods Mol Biol ; 2272: 163-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009613

RESUMO

5-Methylcytosine (5mC) is one of the most abundant and well-studied chemical DNA modifications of vertebrate genomes. 5mC plays an essential role in genome regulation including: silencing of retroelements, X chromosome inactivation, and heterochromatin stability. Furthermore, 5mC shapes the activity of cis-regulatory elements crucial for cell fate determination. TET enzymes can oxidize 5mC to form 5-hydroxymethylcytosine (5hmC), thereby adding an additional layer of complexity to the DNA methylation landscape dynamics. The advent of techniques enabling genome-wide 5hmC profiling provided critical insights into its genomic distribution, scope, and function. These methods include immunoprecipitation, chemical labeling and capture-based approaches, as well as single-nucleotide 5hmC profiling techniques such as TET-assisted bisulfite sequencing (TAB-seq) and APOBEC-coupled epigenetic sequencing (ACE-seq). Here we provide a detailed protocol for computational analysis required for the genomic alignment of TAB-seq and ACE-seq data, 5hmC calling, and statistical analysis.


Assuntos
5-Metilcitosina/análogos & derivados , Metilação de DNA , DNA/análise , DNA/química , Genoma Humano , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sulfitos/química , 5-Metilcitosina/química , Biologia Computacional/métodos , DNA/genética , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Oxirredução
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